The autonomously replicating parvoviruses contain linear single-stranded DNA genomes of about 5 kilo bases. They are widespread and infect invertebrates, birds, and mammals including man. The parvovirus nonstructural protein NSI has a central role in regulating parvovirus replication. NS1 has been assigned 6 phenotypes: 1. positive regulation of the coat protein promoter, 2. negative regulation of its own gene and heterologous gene expression, 3. requirement for parvovirus DNA replication, 4. requirement for excision if viral termini from a plasmid, 5. inhibition of SV40 DNA replication, and 6. inhibition of cell DNA replication. Our evidence suggests that all or nearly all parvovirus DNA replication requires DNA polymerase delta. We propose to use site-specific mutagenesis to study the roles of NSI in viral DNA replication and its interaction with pol delta. Parvovirus DNA replication is restricted in normal human fibroblasts and this restriction is relieved by neoplastic transformation. The defect in normal cells is at the level of viral DNA replication and it cannot be ascribed to a deficiency in viral] gene expression, rather it appears to result from a deficiency in nuclear translocation of NSI. This project will study the functions of DNA polymerase delta and NSI in viral and cellular DNA replication and explore the defect in viral DNA replication between normal and neoplastically transformed cells.